Vaccine strategies that elicit robust mucosal immunity may potentially be effective in preventing mucosal transmission of HIV and other pathogens. We will develop Streptococcus mitis, as a potential vaccine vector for the induction of protective mucosal immunity against HIV. S. mitis has important features that makes it an attractive mucosal vaccine vector. It is a commensal nonpathogenic bacterium in pediatric and adult populations and it is genetically tractable for expression of vaccine immunogens. The organism is most abundant in the mouth and efficiently colonizes the throat and nasopharynx and this microbe has been shown to induce durable mucosal immunity. Importantly, this oral bacterium can be delivered easily and safely via the oral route. The main objective of this proposal is to exploit these advantages to generate a safe recombinant oral vector that will effectively induce anti-HIV mucosal immune responses. We have generated a stable recombinant S. mitis expressing a HIV/SIV and we found that oral delivery of this vaccine vector was safe and in a heterologous rSmitis prime attenuated viral vector/Env protein induced robust mucosal and systemic cytotoxic T cell (CTL) response and neutralizing antibody response. In this proposal, we will further develop rS. mitis vaccine vectors by performing extensive immunogenicity studies, preclinical safety assessments and determine bacterial factors that impact immunogenicity with the goal of refining the vaccine vector. The Thai RV144 human trial and current preclinical studies show that the most promising vaccine strategies may involve combining various vaccine delivery systems with different immunogenic properties in a heterologous prime- boost strategy that utilizes HIV/SIV envelope protein immunogens and pox or adenoviral vectors. Therefore, we will develop a heterologous rSmitis-prime rMVA and Env protein boost vaccine strategy. The central hypothesis is that our heterologous prime-boost strategy will induce high magnitude and durable mucosal and systemic CTLs and neutralizing antibodies against HIV-1.The Specific Aims of this project are: 1) Conduct preclinical immunogenicity assessment of rSmitis prime- recombinant MVA vector and Env protein boost strategy in small laboratory animals. 2) Determine the role of bacterial factors on anti-HIV/SIV mucosal immune responses with the goal of fine-tuning vector immunogenicity. 3) Conduct preclinical immunogenicity assessment of rSmitis prime- recombinant MVA vector and Env protein boost strategy in non-human primates. There is a critical need for an effective HIV vaccine and the proposed studies could lead to the development of a novel and innovative rSmitis-based vaccine strategy as a mucosal AIDS vaccine. Promising findings from our preclinical immunogenicity studies would warrant further preclinical efficacy trials in nonhuman primates, which is a critical step towards human trials.